Compositions and methods for treating bladder cancer

ABSTRACT

The present invention provides methods and compositions for treating bladder cancer. In particular, the present invention provides a fusion protein comprising a toxin moiety that is linked to an epithelial growth factor (EGF) moiety. The toxin moiety and the EGF moiety can be linked optionally via a linker. Typically, the fusion protein is administered intravesically into the cancerous bladder.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority benefit of U.S. ProvisionalApplication No. 61/251,676, filed Oct. 14, 2009, which is incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to treating bladder cancer using a fusionprotein comprising a toxin moiety that is linked to an epithelial growthfactor (EGF) moiety, optionally via a linker. Typically, the fusionprotein is administered intravesically into the cancerous bladder.

BACKGROUND OF THE INVENTION

Bladder cancer is a common cancer with an estimated 67,160 new cases and13,750 deaths in 2007. Most patients with non-muscle-invasive(superficial) cancers are initially treated with cystoscopic resectionsometimes followed by intravesical therapy with bacillus Calmette-Guerin(BCG) solution. This solution contains live, weakened bacteria thatstimulate the immune system to kill cancer cells in the bladder. Thedoctor will typically use a catheter to put the BCG solution in thebladder, and the patient needs to hold the solution in the bladder forat least about two hours. BCG bladder cancer treatment is usually doneonce a week for six weeks. BCG is a non-specific and irritating agentthat has been in use for more than 30 years with little change. BCGlacks acceptable efficacy and has many side effects and limitedtolerability. Some of the side-effects of BCG treatment include, but arenot limited to, irritation of the bladder; an urgent need to urinate;the need to urinate frequently; pain, especially when urinating;fatigue; blood in the urine; nausea; a low-grade fever; and chills.

Moreover, many patients with non-invasive bladder cancer have arecurrence, with a recent meta-analysis reporting a recurrence rate of39% after BCG therapy. In patients with high risk non-invasive bladdercancer, recurrence after intravesical BCG is very common with arecurrence rate in excess of 50%.

Accordingly, there is a need for effective non-invasive methods fortreating bladder cancer.

SUMMARY OF THE INVENTION

Some aspects of the invention provide methods for treating bladdercancer in a subject. Such methods generally comprise administering adiphtheria toxin epidermal growth factor (DT-EGF) fusion proteindirectly to a cancerous bladder of the subject. Often the DT-EGF fusionprotein is administered directly to cancerous cells of the bladder.Typically, the DT-EGF fusion protein is administered intravesically(i.e., directly instilled) into the cancerous bladder. The bladder iseasily accessible via a fiberoptic cystoscope. For example, after thediagnosis of superficial bladder cancer, patients are regularlyre-examined by cystoscope every 3-6 months for the first few years.Biopsies are routinely obtained via the cytsoscopy. Such methods can beused to instill DT-EGF fusion protein into the cancerous bladder.

In some embodiments, the amount of DT-EGF fusion protein administeredranges from about 500 ng/mL to about 2,000 ng/mL, and typically fromabout 500 ng/mL to about 1,500 ng/mL.

Methods for producing DT-EGF fusion proteins are known to one skilled inthe art. However, most conventional methods utilize E. coli to produceDT-EGF fusion proteins, which can be difficult and results in DT-EGFfusion proteins that have limited stability. The Present inventors havefound that many problems associated with using E. coli to produce DT-EGFfusion proteins, including limited stability of the resulting DT-EGFfusion protein, can be avoided by using Pichia pastoris. In order toproduce DT-EGF in P. pastoris, the DNA encoding DT-EGF was modified to(a) introduce an N-terminal alanine, (b) optimize codon usage forefficient translation in P. pastoris, (c) abolish N-linked glycosylationsites, (d) optionally add a linker (e.g., (G₄S)₃ or G₁₀, where G isglycine and S is serine) between the DT and EGF moieties, and (e) addrestriction sites for subcloning in the pPICZalpha yeast expressionplasmid containing an alpha factor prepro leader sequence and the AOX1promoter.

Some aspects of the invention thus provide DT-EGF fusion proteinproduced by P. pastoris using the modified DNA described herein as wellas P. pastoris transfected with the modified DNA, vector comprising themodified DNA, and the modified DNA itself.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the percentages of surviving bladder cancercells 72 hours after treatment with DT-EGF fusion protein at variousconcentrations.

FIG. 2 is a graph showing the results of the specificity of DT-EGF forcells that express the receptor of EGF.

FIG. 3 shows the effect of DT-EGF to suppress the formation of colony ofbladder cancer (clonogenic assay) utilizing a treatment or exposure timeof 2 hours. Panel A shows bladder cancer cell line HT1376 colonizationwith no treatment after 2 hours. Panel B shows the result of treatingthe same with 25 ng/mL of DT-EGF after 2 hours. Panel C shows the resultof bladder cancer cell colonization at 24 hours with no treatment. PaneD shows the result of bladder cancer cell colonization at 24 hours aftertreatment with 25 ng/mL of DT-EGF.

FIG. 4 shows results of not treating and treating mice that wereimplanted with bladder cancer cells. Panel A shows the mice seven daysafter having been implanted with human bladder cancer cells. Panel Bshows the mice two weeks after first DT-EGF treatment (two mice on theright), and DT-GMCSF treated mice (two mice on the left).

FIG. 5 shows the results of cytotoxicity assay on U373MG cells after 50h incubation with dilutions of Bacterial DT-EGF (bDT-EGF) or PichiaDT-EGF (pDT-EGF). One microcurie 3H-thymidine added for 18 h thenharvest on glass fiber mats and counted in LKB Betaplate reader.□-bDT-EGF (IC₅₀=4.7 μM). ▪-pDT-EGF (IC₅₀=1.4 μM).

DETAILED DESCRIPTION OF THE INVENTION

Targeted toxins for use in chemotherapy are fusion proteins that combinea targeting molecule that selectively binds to and enters tumor cellswith a toxin that kills the target cells. Clinical trials of targetedtoxins directed against various tumors have led to FDA approval ofdenileukin diftitox (ONTAK), which is a fusion of diphtheria toxin (DT)and interleukin-2 (IL-2), for the treatment of cutaneous T celllymphoma. Targeting is typically achieved with antibodies or growthfactors that bind to tumor cell receptors. Toxins are often derived frombacterial pathogens (e.g. diphtheria toxin) or plants.

Epithelia growth factor receptor (EGFR) plays an important role inbladder cancer pathogenesis. It has been shown that bladder cancer cellsexpress EGFR protein. In contrast, EGFR is quite uncommon in the normal(i.e., non-cancerous) bladder epithelial cells. Accordingly, the presentinventors have discovered that bladder cancer can be effectively treatedusing a fusion protein comprising a toxin that is linked to anepithelial growth factor protein (EGF). In this manner, the selectivebinding of EGF by cancerous bladder epithelial cells allow selectiveadministration of the toxin to cancerous bladder cells. The fusionproteins of the invention comprise a toxin moiety (e.g., DT toxin)linked to an EGF moiety (targeting moiety). In some embodiments, thetoxin moiety is linked to the EGF moiety through a linker. Thus, someaspects of the invention provide a fusion protein where both toxin andEGF domains are produced from a recombinant construct. As theapplication provides the necessary information regarding the arrangementof toxin and antibody domains, and the sub regions within them, it willbe recognized that any number or chemical coupling or recombinant DNAmethods can be used to generate a fusion protein of the invention. Thus,reference to any particular fusion protein or a coupled toxin-EGF is notnecessarily limiting.

The present invention will now be described in reference to diphtheriatoxin (DT)-EGF fusion protein and methods for using the same to treatbladder cancer. However, it should be appreciated the scope of theinvention is not limited to DT-EGF fusion protein as other toxinmoieties can be used instead of DT moiety.

The DT-EGF fusion protein can be a fusion protein producedrecombinantly. Alternatively, DT moiety and EGF moiety can beindependently recombinantly produce and linked via chemically.Typically, however, DT-EGF fusion protein is recombinantly produced. TheDT toxin moiety can be a truncated mutant or a wild type moiety, such asDT₃₉₀, DT₃₈₉, DT₃₈₃, DT₃₇₀, DT₃₈₈, or other truncated mutants, with andwithout point mutations or substitutions, as well as a full length toxinwith point mutations. Thus, as just illustrative examples of the DT-EGFfusion protein, the invention provides DT₃₈₉-EGF, and DT₃₉₀-EGF fusionproteins. Derivatives of these fusion proteins can be designed andconstructed by one skilled in the art given the disclosure of thepresent application.

The toxin moiety retains its toxic function. Often the toxin moiety alsoretains its membrane translocation function to the cytosol in fullamounts. The loss in binding function located in the receptor bindingdomain of the protein (i.e., DT) diminishes systemic toxicity byreducing binding to non-target cells. Thus, the fusion proteins of theinvention can be selectively and relatively safely administered. Therouting function normally supplied by the toxin binding function issupplied by the EGF moiety. It should be appreciated that the EGF moietyincludes at least a portion of the epitope that is recognized by EGFR ofthe bladder cancer cells.

Any EGF moiety that can be selectively recognized by the bladder cancercells is effective with the toxin moiety, provided that the toxinachieves adequate proteolytic processing along this route. Adequateprocessing can be determined by the level of cell killing.

The recombinant fusion proteins of the invention can be produced fromrecombinant EGF moiety and recombinant DT moiety each containing asingle unpaired cysteine residue. An advantage of this method is that DTmoiety and EGF moiety are easily produced and properly folded using avariety of bacteria or other cells. For chemically coupled DT-EGF fusionprotein, one or more amino acids (e.g., glycine, serine, or anycombination thereof) can is inserted between the DT moiety and the EGFmoiety. However, typically the fusion proteins of the invention areproduced recombinantly. Such recombinant production of the fusionproteins of the invention can include optionally inserting a linker bymodifying the plasmid construct that is used for recombinant productionof fusion proteins.

Some methods of the invention use DAB₃₈₉EGF. DAB₃₈₉EGF is a toxic fusionprotein of diphtheria toxin and the epidermal growth factor (EGF) inwhich the domain recognized by the diphtheria toxin (DT) receptor (e.g.,DT domain amino acids 391-535) is replaced with EGF, so that thetargeted toxin is recognized by the native epidermal growth factorreceptor (EGFR). Superficial bladder cancer commonly expresses EGFRwhereas the normal bladder lining does not. Superficial bladder cancerfrequently recurs after initial treatment, with these recurrent casespotentially leading to cystectomy (surgical removal of the bladder) oradvanced disease. Some methods of the invention utilize theaccessibility of superficial bladder cancer to direct treatment. Suchmethods include instillation of DT-EGF into the bladder to selectivelytarget cancerous bladder cells.

Additional objects, advantages, and novel features of this inventionwill become apparent to those skilled in the art upon examination of thefollowing examples thereof, which are not intended to be limiting. Inthe Examples, procedures that are constructively reduced to practice aredescribed in the present tense, and procedures that have been carriedout in the laboratory are set forth in the past tense.

EXAMPLES

DT-EGF was initially produced by E. coli. However the production in E.coli was difficult to prepare and had limited stability. Repeatedefforts to standardize refolding from bacterial inclusions gave pooryields and purity. To overcome these problems, DT-resistant proprietaryPichia pastoris strains and modified gene expression plasmids was used.In order to produce DT-EGF in P. pastoris, the DNA encoding DT-EGF wasmodified to (a) introduce an N-terminal alanine, (b) optimize codonusage for efficient translation in P. pastoris, (c) abolish N-linkedglycosylation sites, and optionally (d) add a (G₄S)₃ or G₁₀ (where G isglycine and S is serine) linker between the DT and EGF moieties. Theplasmid DNA was transformed into a DT-resistant P. pastoris strain(JW107: a wild type DT resistant strain; Mut⁺, DT^(R), Ura3⁺, His4⁺).Using this transformant, fermentation and purification procedures weredeveloped. After 24 hr induction, an average expression level of 18.5mg/L was obtained. DT-EGF in culture supernatant was purified by aseries of chromatography such as (1) Butyl-650M hydrophobic interactioncolumn step, (2) Poros 50 HQ anion exchange column step, (3) Phenyl HPhydrophobic interaction column step, (4) Butyl-650M hydrophobicinteraction column step, and (5) Q HP anion exchange column step. Itsfinal yield was 30% (5.6 mg/L). The final preparation had >98% ofpurity, <0.04% aggregates, and 6.0 μM of IC50 to an EGFR1 bladder cancercell line.

DT-EGF Gene Construction

An intermediate vector named “A-dmDTop390-bisFv(G4S) in pET” and thepnPICZalpha vector (Woo et al., Protein Expr Purif., 2002, 25, 270-282,which is incorporated herein by reference in its entirety) were used toconstruct an expression vector having the DT-EGF gene.

The EGF gene was amplified using two primers, 5′NcoI-EGF(5′-TTCTTGCCATGGAACAGCGATAGCGAATGCCCG-3′ SEQ ID NO: 1) and 3′E-st-EGF(5′-CGTGAATTCTTAGCGCAGTTCCCACCATTTCAG-3′ SEQ ID NO: 2). PCR wasperformed for 30 cycles at the following conditions: 1 cycle at 94° C.for 2 min, 30 cycles of 94° C. for 30 sec followed by 55° C. for 30 secand then 72° C. for 80 sec, and 1 cycle of 72° C. for 7 min. PCRproducts were analyzed by 1% agarose gel electrophoresis containing 40μg of ethidium bromide (EtBr, BioRad) per 100 mL of 1% agarose (Gibco).DNA fragments with correct size were excised from the agarose gel andpurified using Qiagen gel extraction kit.

The amplified EGF gene was digested with NcoI and EcoRI restrictionenzymes. The intermediate vector was also digested with NcoI, EcoRI andcalf intestine phosphatase. A large DNA fragment of ˜4 kbp was isolatedand purified. The large DNA fragment and the digested EGF gene wereligated and transformed into E. coli NovaBlue competent cells. A newconstruct named “A-dmDTop390-bisFv(G4S)-EGF in pET” was obtained.

The above new construct was digested with XhoI and EcoRI enzymes inorder to obtain a DNA fragment, “X-A-DT390-EGF-E”. The X-A-DT390-EGF-Efragment was inserted between XhoI and EcoRI sites of the pnPICZalphavector. The resulting vector, “X-A-DT390-EGF-E in pnPICZalpha” wastransformed into a DT resistant strain (JW107). Ten μg of plasmid DNAwas linearized with Sad and then electroporated into the JW107 strainutilizing 20052, 7500V/cm and 25 μF with a Bio-Rad GenePulser (Bio-RadLaboratories). Transformants expressing zeocin markers were obtained byspreading onto the zeocin agar plate. Six single colonies from eachtransformation were grown in 5 ml of YPD medium (1% yeast extract, 2%peptone and 2% dextrose) in 14 ml test tubes for 2 days to obtain asaturated cell density and then resuspended in 3 ml of BMMYC medium (1%yeast extract, 2% peptone, 100 mM potassium phosphate, pH 7.0, 1.34%yeast nitrogen base without amino acids, 4×10⁻⁵% biotin, 0.5% methanoland 1% casamino acids) for induction. About 15 μL of methanol wassupplemented every 24 hours after initiation of methanol induction. Thesupernatants were harvested after 2 days of methanol induction and thensubjected to SDS-PAGE or Western blotting to check the DT-EGF expressionlevel. Finally, DT-EGF expression Pichia pastoris strain was obtained.This DT-EGF fusion protein has an N-terminal alanine; no N-linkedglycosylation site and a restriction site for subcloning in the pnPICZayeast expression plasmid containing an alpha factor prepro leadersequence and the AOX1 promoter.

Fermentation

In some instances, the expression of pDT-EGF (i.e., expression of DT-EGFby P. pastoris) was optimized by tuning fermentation procedure below.Pichia pastoris transformed with pnPICZalpha-DT-EGF was maintained asfrozen stocks at −80° C. in 25% (w/v) glycerol. One mL of frozen stockswas inoculated into 50 mL YSG broth (1% yeast extract, 2% soytonepeptone, 2% glycerol) and cultivated for two days at 28° C. at 250 rpm.Twelve mL from these cultures were inoculated into 250 mL YSG broth in a1-L flask and cultured for one day at 28° C. at 250 rpm. Cultures werethen be used as the second seed cultures for inoculation of 4 L ofcomplex fermentation medium—2% yeast extract, 2% soytone peptone, 4%glycerol, 1.34% yeast nitrogen base with ammonium sulfate but withoutamino acids (Difco), and 0.04% foam away (Invitrogen). The fermentationswere done in a BioFlo 110 fermentor (New Brunswick Scientific) with amethanol sensor and controller (Raven Biotechnology) to maintainmethanol at set concentrations during induction. The fermentor waslinked to a computer running AFS BioCommand Windows-based software.After consumption of glycerol detected by a spike in dissolved oxygen,there was a 75% glycerol feed for 7 hours (0.1 to 3 grams/min) and thenmethanol induction controlled to maintain methanol at 0.15%. Dissolvedoxygen was maintained at >25% by adding oxygen as needed. The pH wasmaintained at 6.5 during fermentation by adding 29% NH₄OH or 40% H₃PO4as needed. Temperature was set at 28° C. for growth and ramped down to15° C. during the first four hours of methanol induction and thenmaintained at 15° C. Optimal induction time was 24 hours. After 24 hrinduction, an average expression level of 18.5 mg/L was obtained.

Purification

Purification procedure was developed to obtain DT-EGF with >98% purity.The purification procedure includes (1) Butyl-650M hydrophobicinteraction column step, (2) Poros 50 HQ anion exchange column step, (3)Phenyl HP hydrophobic interaction column step, (4) Butyl-650Mhydrophobic interaction column step, and (5) Q HP anion exchange columnstep. Its final yield was 30% (5.6 mg/L).

Characterization of the DT-EGF Preparation

In order to characterize the DT-EGF preparations during processdevelopment and confirm the feasibility of the tuned productionprocedure, the following procedures were performed: (a) proteinaggregate analysis by size exclusion, (b) SDS-PAGE analysis, (c)measurement of DT-EGF concentration, (d) potency assay, and (e)endotoxin assay test. The final preparation had >98% of purity, <0.04%aggregates, 6.0 μM of IC50 to an EGFR1 bladder cancer cell line and <5.0EU/mg of DT-EGF.

In Vivo Tests of DT-EGF Treatment

Human bladder cancer cells (HTB9) were infected with lentiviruscontaining the firefly luciferase gene (HTB9-luc), allowing for thenon-invasive monitoring of the implanted cells. For tumor implantation,6-8 week old female nu/nu mice were anesthetized and their bladdercatheterized with a 24 gauge plastic catheter, instilling 100 μL of1.5×10⁶ HTB9-luc cells. The cells were held in the bladder for 3 hoursby a retention suture temporarily placed around the urethra. Treatmentwas pursued only in mice with implantation of the HTB9-luc cellsconfirmed by the presence of luciferase activity 1 week after theimplantation procedure. DAB₃₈₉EGF was given in the active treatmentgroup and DAB₃₈₈GMCSF in the control arm, with 70 ng of drugadministered twice weekly for 2 weeks in 70 μL of solution. The drug wasretained in the bladder for 2 hours in an analogous manner to theimplantation procedure, using a temporary retention suture underanesthesia. A total of 6 mice were treated in each group. In thosereceiving DAB₃₈₉EGF, 5 of 6 had complete loss of luciferase activityafter 2 weeks of intravesical therapy. One of these 6 mice had reductionin luciferase activity, but a low level of residual activity persistedafter the 2 week treatment period. In contrast, luciferase activitypersisted in 5 of 6 mice in the DAB₃₈₈GMCSF control arm at 2 weeks.

Results and Discussion

The present inventors have discovered and demonstrated effectiveness ofDT-EGF in treating bladder cancer. The activity of DT-EGF was assessedin vitro to determine if exposing bladder cancer cells to DT-EGF wouldbe lethal. As shown in FIG. 1, more than half of the bladder cancercells were killed or suppressed when exposed to DT-EGF for 72 hours. Thespecificity of DT-EGF was also assessed. While many clinically usedtargeted agents in oncology require strong reliance to a particularmolecular pathway to be effective, without being bound by any theory itis believed that DT-EGF merely requires a threshold level of intact EGFreceptors to be effective. When the receptor is present, it allowsDT-EGF to enter the cell where the DT or diphtheria toxin portion killsthe cells. To test the specificity of DT-EGF for cells that express thereceptor of EGF, a lung cancer cell line (H520) that does not expressEGF receptor was included along with the bladder cancer cells. As shownin FIG. 2, H520 was not affected by DT-EGF at doses 5-10 times theeffective dose in multiple tested bladder cancer cell lines (note the Xaxis is logarithmic, to capture this large dose range). Without beingbound by any theory, it is believed that this result is indicative thatDT-EGF was exerting a specific killing action based on the presence ofthe EGF receptor, rather than a non-specific toxicity to any cell.

The practical considerations of delivering this drug to patients withbladder cancer was considered. Generally, it is difficult for patientsto “hold in” intravesical agents in the bladder for more than 1-2 hours.Consider that in addition to the volume of DT-EGF or other therapeuticagent instilled in the human bladder, the kidneys continue to produceurine and fill up the bladder. Therefore, the effect of DT-EGF tosuppress the formation of colony of bladder cancer (clonogenic assay)was determined utilizing a treatment or exposure time of 2 hours. Asshown in FIG. 3, the strong clonogenic suppression (i.e., prevention ofthe formation of new cancer cell colonies) with a single 2 hourtreatment time was clearly observed. This showed that a 1-2 hourtreatment time in humans is suitable.

Animal testing of DT-EGF for bladder cancer was also performed. Thisinvolved a mouse (athymic) bladder cancer model in which the tumor growsin the outer layers of the bladder (orthotopic) and the tumor wasengineered so that its presence can be followed with luciferaseactivity. In the first cohort of mice treated with intravesical DT-EGF,no change in luciferase activity was observed after 1 week; however,there was a loss in luciferase activity in the mice treated with DT-EGFat 2 weeks, but no change in the activity of mice treated with DT-GMCSF,which was used as a control. See FIG. 4. The pathology reviewdemonstrated no noticeable residual tumor in the DT-EGF treated mice,but as expected gross tumor was observed in the DT-GMCSF treated mice.These data showed the efficacy, deliverability, and specificity of thistreatment approach for the treatment of bladder cancer.

As stated above, the present inventors have discovered methods fortreating bladder cancer using DT-EGF. In one particular example, 50 mgof DT-EGF is prepared and provided to bladder cancer subjects. Thepresent inventors prepared DT-EGF in Pichia pastoris because DT-EGFproduced by E. coli was difficult to prepare and had limited stability.Repeated efforts to standardize refolding from bacterial inclusions gavepoor yields and purity. In order to produce DT-EGF in P. pastoris, thepresent inventors have modified the DNA encoding DT-EGF to (a) introducean N-terminal alanine, (b) optimize codon usage for efficienttranslation in P. pastoris, (c) abolish N-linked glycosylation sites,(d) optionally add a linker (e.g., (G₄S)₃ or G₁₀) between the DT and EGFmoieties, and (e) add restriction sites for subcloning in thepnPICZalpha yeast expression plasmid containing an alpha factor preproleader sequence and the AOX1 promoter. A diphtheria toxin resistant P.pastoris strain, JW107 (DT^(R), Mut+, His4+, Ura3+), was transformed andselected on zeocin selection media. Recombinant protein expression wasinduced with methanol and partially purified by hydrophobic interactionand anion exchange chromatography. Protein molecular weight and puritywere evaluated by SDS-PAGE. Cytotoxic potency was improved relative tobacterial DT-EGF (FIG. 5).

The foregoing discussion of the invention has been presented forpurposes of illustration and description. The foregoing is not intendedto limit the invention to the form or forms disclosed herein. Althoughthe description of the invention has included description of one or moreembodiments and certain variations and modifications, other variationsand modifications are within the scope of the invention, e.g., as may bewithin the skill and knowledge of those in the art, after understandingthe present disclosure. It is intended to obtain rights which includealternative embodiments to the extent permitted, including alternate,interchangeable and/or equivalent structures, functions, ranges or stepsto those claimed, whether or not such alternate, interchangeable and/orequivalent structures, functions, ranges or steps are disclosed herein,and without intending to publicly dedicate any patentable subjectmatter.

What is claimed is:
 1. A method for treating bladder cancer in asubject, said method comprising administering a pharmaceuticallyacceptable formulation of a diphtheria toxin-epidermal growth factor(DT-EGF) fusion protein chosen from DT₃₉₀-EGF, DT₃₈₉-EGF, DT₃₈₃-EGF,DT₃₇₀-EGF, and DT₃₈₈-EGF to a subject with bladder cancer and in need ofbladder cancer treatment, wherein the DT-EGF formulation is administeredintravesically and retained in the bladder for approximately 1-2 hours.2. The method of claim 1, wherein the amount of administered DT-EGFfusion protein is about 50 mg.
 3. The method of claim 1, wherein thesubject is a mammal.
 4. The method of claim 3, wherein the subject is ahuman.
 5. The method of claim 1, wherein the bladder cancer issuperficial bladder cancer.
 6. The method of claim 1, wherein the DT-EGFformulation is retained in the bladder for approximately 2 hours.
 7. Themethod of claim 1, wherein the DT-EGF fusion protein of the DT-EGFformulation comprises: an N-terminal alanine; and no N-linkedglycosylation site.